Gc ms solution software package

The raw chromatographic data obtained from the GC–MS analysis (*QDC file) were converted into the ANDI AIA format (*ABF file) from GCMS Solution MS data files using the GC–MS Solution software package (Shimadzu, Kyoto, Japan). Peak alignment, filtering, and annotations were completed using MS-DIAL (Riken, Tokyo, Japan). In the annotation step, the QC sample acted as a reference. The metabolites were tentatively annotated according to their RIs recorded on RI GL-Science DB (InertCap 5MS-NP, predicted Fiehn RI, 494 records), downloadable from the MS-DIAL official website. Metabolite peaks were considered if the height was five times higher than the blank (see Supplementary Materials). Furthermore, additional filtering was applied by selecting data that showed a relative standard deviation (RSD) of less than 30% within the QC samples. Tentatively annotated metabolites were subjected to PCA using the commercial software SIMCA P+ ver. 13.0.3 package (Umetrics, Umea, Sweden) [11 (link),21 (link)].

The raw chromatographic data were converted into ANDI files (Analytical Data Interchange Protocol,*.cdf) using GC-MS Solution software package (Shimadzu). The data were imported to MetAlign software [25] (link), [26] (link) (Wageningen UR, The Netherlands, available for free at the website http://www.pri.wur.nl/UK/products/MetAlign/) for peak selection and alignment. The peak intensity of each compound was normalized based on the ribitol internal standard. AIoutput2 (version 1.29) was used as annotation software. The retention indices of all detected metabolites were calculated based on the standard alkane mixture and tentative identification of metabolites was done by comparing the retention indices with our in-house library [27] (link) to aid the tentative identification of compounds. On the other hand, the retention time of each metabolite was used to compare with the NIST 2011 Library (NIST11/2011/EPA/NIH).

Chromatographic GC–MS analysis data were converted into the netCDF format using the GC–MS Solution software package (Shimadzu, Kyoto, Japan) following previously published methods [20 (link),21 (link)]. This included file format conversion to analytical data interchange protocol (ACDF, CDF). Peak alignment detection, baseline correction, and alignments were performed using the freely available software package MetAlign and Output version 1.30, respectively. The pooled QC data were utilized in MetAlign as reference data. The processed data were then exported to the CSV-format file. Peak RIs were calculated on the basis of the retention time of the standard alkane mixture. By comparing the RIs and their mass spectra with an in-house library prepared from authentic standards, tentative identifications were performed using AIoutput2 annotation software, and the data matrix was constructed. The peaks which were not of biological origin were excluded manually from the data matrix (refer to the chromatograph of the blank). The mass spectra of all the peaks were compared with the NIST and Wiley libraries, and the retention times and the mass spectra of sugar peaks were compared with the authentic standards to confirm the tentative identifications. The assigned peak intensities were normalized against the intensity of the ribitol internal standard.